Effects of a Hexachlorobiphenyl and Pentachlorophenol on Growth and Photosynthesis of Phytoplankton

J. Great Lakes Res. 8(2): 328-33 5 Internat . Assoc. Great Lakes Res. 1982

EFFEC TS OF A HEXA CHLO ROBIP HENY L AND PENT ACHL OROP HENO L ON GROW TH AND PHOT OSYN THES IS OF PHYT OPLA NKTO N

Ivan J. Gotham and G-Yull Rhee 1 Environmental Health Institute Division of Laboratories and Research New York State Department of Health Albany, N. Y. 12201

ABSTR ACT. The effects of two organochlorine compounds 2,4,5,2',4',5' hexachlorobiphenyl (HCB) and pentachlorophenol (PCP) on photosynthesis and growth were investigated in semicontinuous cultures of three species of algae: Ankistrodesmus falcatus, Melosi ra sp., and Photosynthesis appeared to be stimulated by HCB in A. falcatus and Melosir Microcystis sp. a sp., both per cell and per unit of chlorophyll, at average cellular HCB concentrations ofappr oximately 3 x 10-5 and 6 x 10-6 I ng cell- respectively. At the same HCB concentrations the growth rate of Melosira was significantly inhibited, but that of A. falcatus showed no significant trend. PCP appeared to inhibit photosynthesis in A. falcatus and Microcystis sp., both per cell and per unit ofchlorophyll, at a cell PCP concentration range ofapproximately 4-8 x 10-7 and 2-11 x 10-8 ng cell-I, respectively. In Melosira, photosynthesis per unit ofchlorophyll was inhibited at 2-6 x 10- 7 ng cell-I. At these PCP concentrations, only Melosira showed a discern ible decrease in growth rate.

INTRO DUCT ION The wide distribution of organochlorine chemicals in the Great Lakes is well documented (e.g., Konasewitch et al. ·1978, Holdrinet et al. 1978, Frank et al. 1979 and 1980, Strach an and Huneault 1979, Simmons et al. 1980). As these compounds are highly lipophiliC, they tend to partiti on strongly into organic particulates including planktonic algae. Most of these chemicals are toxicants to phytoplankton, but their effects on photosynthesis and growth rate in algae vary widely between species, ranging from inhibition through no effect to stimulation (e.g., Ukeles 1962, Wurster 1968, Vance and Drumm ond 1969, Menzel et al. 1970, Mosser et al. 1972, Luard 1973, Cole and Plapp 1974, Craigie and Hutzinger 1975, Glooschenko and Glooschenko 1975, Fisher 1975, Walsh et al. 1977, Harding and Phillips 1978). The type and magnitude of responses by organisms may vary, not only with species and with the kind and the dose of chemicals, but also with other factors in the experimental design. One of the more I

impor tant factors is popula tion density relative to the concentration of the toxicant added. Thus in batch cultures, where populations increase with time, measurement of effects with respect to one initial dose can be misleading, as the dose per cell decreases as the popula tion density increases. Moreover, in most earlier studies the distribution or mass balance of toxicants in the cultures was not determined. Therefore, it is difficult to make a quantitative interpretation of the results. In an attemp t to better understand the effects of organochlorine contaminants in more quantitative terms, the influence of the two chlorinated compounds, 2,4,5,2'4',5'-hexachlorobiphenyl (HCB) and pentachlorophenol (PCP) , on photosynthesis and growth was examined in three species of algae isolated from the Great Lakes in semicontinuous culture. These compounds have been widely detected in the water and fishes {)f the Great Lakes (Konasewich et al. 1978; G. Veith, personal communication). The semicontinuous culture technique permits a regular supply of toxicants and fresh medium to the culture, which obviates the shortcomings of the batch culture system.

Address for communication and reprint requests.

328

ORGANOCHLORINE CHEMICALS AND PHYTOPLANKTON MATERIALS AND METHODS The three test species, Ankistrodesmus falcatus, Melosira sp., and Microcystis sp., were isolated in our laboratory from waters of Saginaw Bay, Lake Huron. Semicontinuous axenic cultures were grown in a nutrient-sufficient, chemically-defined medium (Rhee and Gotham 1981) with the test chemical HCB or PCP. Parallel control cultures were grown without the chemicals. All cultures were grown under continuous illumination of about 17 W'm 2 at 20 0 C. The semicontinuous culture vessels were made from 4-L Pyrex glass bottles each fitted with two glass siphon tubes and an aeration port plugged with loose glasswool. The siphon tubes were used to remove cell suspensions and to add the same volume of fresh medium. The cultures were mixed constantly with a Tephlon-coated magnetic stirring bar. Each day 1 L of the 3-Lculture volume was replaced with fresh medium. Growth rate (f.L) was calculated as

where D is dilution rate and XI and X2 are cell numbers at times tl and t2. 14C-labelled PCP (97% purity) and HCB (99%) were obtained from KOR Laboratories Inc. (Cambridge, Mass.) and New England Nuclear (Boston, Mass.), repectively. Preparation of the HCB medium without organic carrier solvents is described elsewhere (Lederman and Rhee 1981a). The concentration of HCB in the reservoir medium ranged from 0.5 to 1.0 f.Lg . L-I. As PCP has an aqueous solubility of 20 mg . L-I (Firestone 1977), it was solubilized directly into the reservoir medium to give final reservoir concentrations of 1.0 to 1.3 f.Lg . L-I. Both control and treated cultures were monitored daily for population growth (cell number), photosynthetic rate (14C uptake), extracted chlorophyll fluorescence, and the concentrations of test chemicals in the total culture volume, cells, and aqueous phase. The concentrations of the test chemicals were quantified from their specific activities on a scintillation spectrometer (Beckman). Chlorophyll determinations and other analytical methods have been described elsewhere (Lederman and Rhee 1981a,b). The relative response of the treated cultures was calculated as response/ cell (treated) - response/ cell (control) response/ cell (control)

329

Significance tests for stimulation or depression relative to controls were conducted on all data points except those corresponding to 1 h after addition of toxicant to cultures. The nonparammetric test used in this study was that of Wilcoxon's signed ranks. RESULTS Hexachlorobiphenyl With the addition of HCB, Melosira sp. and A. falcatus showed an initial decrease in photosynthetic rate per cell relative to controls (Figures 1,2). However, after the second or third day of incubation they exhibited a recovery or stimulation relative to control at P < 0.01 and P < 0.10, respectively. Such responses also occurred when photosynthesis was expressed per unit of cellular chlorophyll and the trend of stimulation was significant at P < 0.05 in both species. It could not be determined whether this change in photosynthesis was related to cellular HCB because of the scatter in the data. The effects of HCB on growth rate (Figures 1,2) appear to be different from those on photosynthesis. In Melosira sp. growth rate decreased gradually the first 2 days accompanied by an increase in cellular HCB, and then remained at a generally constant low rate until the eighth day. This relatively constant growth rate was accompanied by little change in cellular HCB. The overall decrease in growth rate during the experimental period is significant a P < 0.01. Although a general dose-response relationship has been observed in Fragilaria crotonensis (Lederman and Rhee 1981 b), it is not clear whether changes in growth rate and cellular HCB observed here in Melosira sp. indicate a similar dose-response relationship because of the scatter in the growth rate measurement. In A. falcatus, on the other hand, growth rate appears to have no relationship to cellular HCB, as growth rate appears to have increased despite the accumulation of HCB from the second to fourth day of incubation. The data for A. falcatus were not sufficient to demonstrate a clear trend in relative stimulation. The growth rate of control cultures remained generally constant during the experimental period. The average growth rates (mean ± SE) were 0.72 ± 0.05 day-I for A. falcatus, 0.67 ± 0.04 day-I for Melosira sp., and 0.81 ± 0.06 day-I for Microcystis sp. (Microcystis was only investigated for PCP effects in the present work).

330

GOTHAM and RHEE

In Melosira cellular chlorophyll a, as measured by extracted fluorescence, was higher m HCB cultures than in controls and showed a generally increasing trend (P < 0.01) with time during the experimental period. In A. falcatus, on the other

hand, there was a decrease during the first 3 days and then a recovery to cellular concentrations generally higher than those in the controls. However, the chlorophyll data were insufficient to demonstrate any clear statistical trend in this species.

EFFECTS OF HCB ON Melosira sp.

EFFECT OF HC B ON Anklslrodesmus folco/us

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FIG. 2. Relative responses of Ankistrodesmus falcatus to HCB expressed as a proportion of the control. The point closest to t = 0 is 1 hr after the toxicant was added.

331

ORGANOCHLORINE CHEMICALS AND PHYTOPLANKTON Pentachlorophenol The kinetics of bioconcentration of PCP was examined in A. faleatus at concentrations ranging between 9 to 22 JJ.g . L-I and at culture densities of 0.4 to 2.1 x 106cells . mL-I. The bioconcentration factor, mg cell PCp· mg cell dry weight- I/ mg PCP mg medium-I, appears ,to be independent of external concentrations but was inversely proportional to the density of the culture (Figure 3). These results are similar to the bioconcentration kinetics of HCB in this species (Lederman and Rhee 1981a) except that the factors for HCB are about two to three orders of magnitude greater than those for PCP.

EFFECT OF PCP ON Ankis/rodesmus folco/us

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HCB, however, the change in growth rate does not appear to be related to the trend of changes in cellular PCP. The data were insufficient to demonstrate any clear statistical trends for A.faleatus and Microeystis sp. (Figures 4,5). In nutrient-sufficient turbidostat and phosphate-limited chemostat cultures of F. crotonensis a decrease of cellular HCB, brought about by stopping the replacement of HCB medium with HCB-free medium in the reservoir, always increased the growth rate, even above the rate 10 control cultures (Lederman and Rhee 198Ib).

332

GOTHAM and RHEE EFFECT OF PCP ON Microcystis sp.

EFFECTS OF PCP ON Melosira sp.

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The effects of PCP on extracted chlorophyll fluorescence varied considerably between species. In Melosira, cellular fluorescence showed an overall increasing trend with time which was significant only at P < 0.10 (Figure 6). A similar increasing trend with time was observed in HCB medium but with no initial depression of fluorescence below the level in controls. In A. falcatus (Figure 4), PCP seems to enhance overall cellular chlorophyll concentrations, but this trend is significant only at P < 0.10. In Microcystis (Figure 5), there is no clear trend.

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DISCUSSION The increase of photosynthetic rate per unit of chlorophyll a in Melosira after HCB exposure was similar to that in phosphate-limited F. crotonensis

ORGANOCHLORINE CHEMICALS AND PHYTOPLANKTON (Lederman and Rhee 1981 b). The inhibition of growth rate was also similar in both species with this toxicant. However, in the latter species there was no increase in photosynthesis per cell. Therefore the increase per unit of chlorophyll a in F. crotonensis was due to a decrease in chlorophyll a content. In Melosira, however, cellular chlorophyll a was higher in HCB-treated cultures relative to control. As growth rate (measured by cell numbers) was inhibited while photosynthetic rate per cell was enhanced by HCB in Melosira, either the cell carbon content or the excretion of organic carbon must have increased as a response to exposure to HCB. A similar increase may have occurred in A. fa Icatus, as carbon assimilation rate per cell increased significantly while growth rate appeared to be generally uncoupled to cellular HCB. An increase in carbon fixation rate on exposure to polychlorinated biphenyls (PCBs) has been observed in situ in natural phytoplankton populations of Lake Huron (D. C. McNaught, personal communication). If stimulation is a common response of planktonic algae to a low concentration of PCBs, contamination of aquatic environments by these chemicals would increase primary productivity or the production of dissolved organic matter, an important limiting substrate for aquatic heterotrophs. It seems, therefore, that the potential impact of pollution by organochlorine compounds must be evaluated in terms, not only of inhibition, but also of their stimulatory effects. The underlying mechanisms of the stimulation of carbon fixation are not clear. However, in Selenastrum capricornutum, DDT increases photorespiration by interrupting cyclic photophosphorylation (Lee et al. 1976). This may enhance the excretion of glycolate or other compounds of the photorespiratory pathway, resulting in a net decrease of photosynthesis. Conversely, a stimulation of carbon fixation may be observed if glycolate synthesis in photorespiration is inhibited by HCB. Our preliminary results indicate that the activity of ribulose biphosphate carboxylase was higher in HCBtreated cells of Melosira than in controls. Diatoms are apparently more sensitive than other groups of algae to toxic organic chemicals. The growth rates of F. crotonensis (Lederman and Rhee 1981 b) and Melosira were significantly decreased by both HCB and PCP, but both toxicants in the green alga A. falcatus and PCP in the bluegreen alga Microcystis sp. had no significant effects even at higher cellular concentrations. There· seems to be a general relationship between

333

the solubility and the bioconcentration factor of HCB and PCP. Aqueous solubility is 1-9 J,Lg • L-I for HCB (Wallenhofer et al. 1973, Haque and Schmedding 1975) and 20 mg . L-I for PCP (Firestone 1977). The bioconcentration factor in A. falcatus is IO L 106 for HCB (Lederman and Rhee 1981a) and 103 for PCP (Figure 3); each factor increases with decreasing culture density. However, PCP appears to be more toxic, as in Melosira a comparable decrease in growth rate was found with HCB at 60 x 10-7 ng . cell- I and with PCP at only 2-6 x 10-7 ng . cell-I. Because of the differences in the bioconcentration factor, there was always much more residual PCP (0.9 - 1 J,Lg' L-I) than HCB (20 - 30 ng . L-I, which was near the limit of detection). Hutchinson et al. (1980) reported a linear relationship between the log values of solubilities and the log of concentrations of hydrocarbons and chlorinated hydrocarbons required to yield 50% inhibition of photosynthesis. In the present study no such relationship can be seen between HCB and PCP. On the contrary, the more soluble toxicant appears to be more toxic. PCP is widely used in the lumber industry as a wood preservative and it is one of the important organic pollutants in the Great Lakes (G. Veith, personal communication). Erickson and Hawkins (1980) reported that, in unialgal cultures and natural populations of marine algae, PCP is toxic at concentrations from 125 to 1 ,000 J,Lg • L-I. The present study shows, however, that this chemical is toxic at concentrations two to three orders of magnitude lower than theirs (Figures 4,5,6). They also found that, for natural populations of marine algae, photosynthesis per unit volume of culture is unaffected by tri- and tetra-chlorophenols at concentrations of 500 to 2,000 J,Lg • L-I. Most previous studies of environmental toxicants were carried out at concentration ranges several orders of magnitude higher than those in natural environments (see references in Introduction). Most of these studies also used batch cultures in which only the initial toxicant concentrations were known. In such studies the effects may vary with time, as any increase in population dilutes the cellular toxicant concentrations; and when the culture density becomes sufficiently high, the toxic effects can be completely abolished. For the same reason, inhibitory effects would be most pronounced during the initial stages of culture. Moreover, the concentrations of toxicants employed were generally much higher than the aqueous solubilities; this was achieved by using carrier solvents

334

GOTHAM and RHEE

such as acetone or alcohol. The control cultures normally contained only the solvents, but it was not determined how these solvents affected the solubility characteristics of the chemicals in water, which may influence their bioconcentration characteristics. Finally, it was not determined whether even trace additions of these solvents altered the membrane permeability of the cells to the toxicants. Bowes (1972) suggested that the resistance of Dunaliel/a tertiolecta may reside at least in part in membrane permeability. In summary, phytoplankton accumulate high concentrations of HCB and PCP from dilute solutions. The bioconcentration factor for PCP is about two to three orders of magnitude less than HCB, probably due to differences in their aqueous solubilities. On a concentration per cell basis, PCP is more inhibitory for photosynthesis than HCB. In fact, HCB was stimulatory for photosynthesis at cellular concentrations two orders of magnitude higher than PCP in both A. falcatus and Melosira sp.

ACKNOWLEDGMENT This work was supported by U.S. Environmental Protection Agency grant R80612610 to GYR. We thank Drs. T. C. Lederman and V. J. Bierman, Jr., for helpful discussions during the course of this work, and Dr. G. W. Fuhs for his comments on the manuscript. Mr. C. Amento provided valuable technical assistance.

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and photosynthesis of marine phytoplankton: A reassessment. Science 189:463-464. Frank, R., Thomas, R. L., Holdrinet, M., Kemp, A. L. W., and Braun, H. E. 1979. Organochlorine insecticides and PCB in surficial sediment (1968) and sediment cores (1976) from Lake Ontario. J. Great Lakes Res. 5:18-27. ____ , , Braun, H. E., Rasper, J., and Dawson, R. 1980. Organochlorine insecticides and PCB in the surficial sediments of Lake Superior (1973). J. Great Lakes Res. 6:113-120. Glooschenko, V., and Glooschenko, W. 1975. Effect of polychlorinated biphenyl compounds on the growth of Great Lakes phytoplankton. Can. J. Bot. 53: 653-659. Haque, R., and Schmedding, D. 1975. A method of measuring the water solubility of hydrophobic chemicals: solubility of five polychlorinated biphenyls. Bull. Environ. Contam. Toxicol. 14:13-18. Harding, L. W., and Phillips, J. H. 1978. Polychlorinated biphenyl (PCB) effects on marine phytoplankton photosynthesis and cell division. Mar. BioI. 49: 93-101. Holdrinet, M. V. H., Frank, R., Thomas, R. L., and Hetling, L. J. 1978. Mirex in the sediments of Lake Ontario. J. Great Lakes Res. 4:69-74. Hutchinson, T. C., Hellebust, J. A., Tam, D., Mackay, D., Mascarenhas, R. A., and Shiu, W. Y. 1980. The correlation of the toxicity to algae of hydrocarbons and halogenated hydrocarbons with their physicalchemical properties, pp. 577-586. In: B. K. Afgan and D. Mackay (Eds.), Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment. Plenum Press, New York. Konasewich, D., Traversy, W., and Zar, H. 1978. Great Lakes water quality. Status report on organic and heavy metal contaminants in the Lakes Erie. Michigan. Huron and Superior Basins. International Joint Commission on Great Lakes Water Quality. Windsor, Ontario. Lederman, T. c., and Rhee, G-Y. 1981a. Bioconcentration of hexachlorobiphenyl in Great Lakes planktonic algae. Can. J. Fish. Aquat. Sci. 39:380-387. _ _ _ _ , and . 1981 b. The influence of a hexachlorobiphenyl on the growth of Great Lakes phytoplankton. Can. J. Fish. Aquat.Sci. 39:388-394. Lee, S. S., Fang, S. c., and Freed, V. H. 1976. Effect of DDT on photosynthesis of Selanastrum capricornutum. Pestic. Biochem. Physiol. 6:46-51. Luard, E. J. 1973. Sensitivity of Dunaliella and Scenedesmus (Chlorophyceae) to chlorinated hydrocarbons. Phycologia 12:29-33. Menzel, D. W., Anderson, J., and Randitke, A. 1970. Marine phytoplankton vary in their response to chlorinated hydrocarbons. Science 167:1724-1726. Mosser, J. L., Fisher, N. S., Teng, T-C., and Wurster, C. F. 1972. Polychlorinated biphenyls: Toxicity to certain phytoplankters. Science 175: 191-192.

ORGANOCHLORINE CHEMICALS AND PHYTOPLANKTON Rhee, G-Y., and Gotham, I. J. 1980. Optimum N:P ratios and coexistence of planktonic algae. J. Phycol. 16:486-489. and . 1981. Effect of environmental factors on phytoplankton growth: Temperature and the interactions of temperature with nutrient limitation. Limnol. Oceanogr. 26:635-648. Simmons, M. S., Bialosky, D. I., and Rossmann, R. 1980. Polychlorinated biphenyl contamination in surficial sediments of northeastern Lake Michigan. J. Great Lakes Res. 6:167-171. Strachan, W. M. J., and Huneault, H. 1979. Polychlorinated biphenyls and organochlorine pesticides in Great Lakes precipitation. J. Great Lakes Res. 5:61-68.

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Ukeles, R. 1962. Growth of pure cultures of marine phytoplankton in the presence of toxicants. Appl. Microbiol. 10:532-537. Vance, B. D., and Drummond, W. 1969. Biological concentration of pesticides by algae. J. Amer. Water Works Assoc. 61:360-362. Wallenhofer, P. R., Koniger, P. R. N., and Hutzinger, O. 1973. Solubilities of twenty-one chlorophenyls in water. Analabs Research Notes 13: 14-17. Walsh, G. E., Ainsworth, K. A., and Faas, L. 1977.. Effects of uptake of chlorinated naphthalenes in marine unicellular algae. Bull. Environ. Contam. Toxicol. 18:297-302. Wurster, C. F. 1968. DDT reduces photosynthesis by marine phytoplankton. Science 159:1474-1475.